Greiner, Erin et al. (2015) International Worm Meeting "Modulation of Transthyretin Degradation Alters Proteotoxicity in Caenorhabditis elegans Models of Degenerative Disease."

Encalada, Sandra, Paulsson, Johan, Dillin, Andrew, Greiner, Erin, Genereux, Joseph, Kelly, Jeffery, Porras-Gonzalez, Diana

[International Worm Meeting, 2015]

The transthyretin (TTR) amyloid diseases, including Familial Amyloid Polyneuropathy (FAP), are the most common autosomal-dominantly inherited systemic amyloidoses, wherein mutant TTR leads to peripheral and autonomic nervous system degeneration, cardiac dysfunction, or both. The tetrameric TTR protein is primarily secreted by the liver and undergoes dissociation and partial monomer denaturation, followed by extracellular TTR aggregation onto distal postmitotic tissues such as the neurons and heart. However, the mechanism(s) of this cell non-autonomous TTR proteotoxicity have not been elucidated in any model organism. To dissect the mechanism(s) of TTR secretion, degradation, and age-dependent aggregation on TTR cell non-autonomous proteotoxicity, we generated and characterized C. elegans transgenic lines expressing human WT TTR, or the destabilized TTR variants V30M or D18G expressed in the body wall muscle under the unc-54 promoter. We find that expressing WT TTR and V30M TTR in the muscle significantly reduced the mean lifespan and increased age-dependent paralysis in these strains. To scrutinize TTR toxicity mechanisms, we are using TTR conformational probes (small molecules and antibodies) to monitor the changes in the levels of TTR conformers generated (i.e., tetramer, misfolded oligomers) throughout the lifespan of these strains in order to formulate TTR structure-proteotoxicity relationships. Using a fluorogenic probe that selectively binds and reacts with TTR tetramers, we show that TTR tetramers are secreted from the muscle in WT TTR and V30M TTR strains and are taken up by coelomocytes, six macrophage-like cells in the body cavity where endocytosis and degradation usually takes place. Genetic ablation of coelomocytes appears to increase V30M TTR tetramer levels in the body cavity, suggesting that TTR degradation occurs in these cells. Moreover, coelomocyte ablation significantly exacerbated V30M TTR proteotoxicity resulting in increased sterility and reduced viability, suggesting that modulation of V30M TTR degradation influences its toxicity. Characterization of these models points toward protein degradation as a relevant factor for modulating toxicity in degenerative diseases of postmitotic tissues.

Akram M. Abou-Zied et al. (2006) European Worm Meeting "Dynamic Expression of Homeobox Genes during C. elegans Embryogenesis"

Johan Koch, David Baillie, Robert Johnsen, Jurgen Hench, Akram M. Abou-Zied, Ding Xue, Thomas R. Burglin

[European Worm Meeting, 2006]

Akram M. Abou-Zied1, Jorgen Hench1, Robert Johnsen2, David Baillie2, Ding Xue3, Johan Koch1, Thomas R. Burglin1 Homeobox genes encode transcription factors of critical importance for development. Systematic studies of dynamic, spatio-temporal gene expression patterns of developmental control genes have been rare so far in C. elegans, We have started to investigate the expression patterns of particular homeobox genes during development using 2-channel 4D microscopy. The PCR-based cloning strategy adapted from Oliver Hobert allows the production of transcriptional fusions in which the upstream regulatory regions of each gene is placed in frame with the translational initiation ATG of the reporter gene GFP. The homeobox::GFP reporter constructs are being used to investigate the expression pattern in larval stages, and more importantly, also to examine the embryonic expression pattern using live GFP time-lapse recordings. We have been recording 4D stacks interspersed with GFP for the following genes ceh-5, ceh-33, ceh-34, ceh-41, ceh-44, and ceh-45, ceh-30, NPax, ceh-6, ceh-32, ceh-26. Additional homeobox genes are in line to be processed. Analysis of our 4D recordings has revealed in many instances early dynamic expression that fades again in later development, whose functional significance is currently unclear. Possibly these events in early development set up patterns, regions and cell fates that are not reflected by the expression seen later in larval stages.. Still, many of the genes start their expression pattern later in embryogenesis. Nevertheless, we find very intriguing expression patterns which show dynamic changes of cell position, incipient expression in a very wide range of cells with no apparent connection, or slowly increasing expression during late embryogenesis. Our recordings reveal a large array of different dynamic strategies that the C. elegans embryo employs to execute its developmental program. To aid in our analysis, we are developing several software tools. Virtual Wormbase (celegans.sh.se) is the major tool on which we are working to ultimately convert GFP expression data into digitized data that can be submitted to Wormbase. We also have developed additional tools to convert our recorded stacks in different formats and produce quicktime movies of the 5 to 10 Gig large datasets that one recording produces. More recently, we also started to increase the image data density (number of slices) to allow better lineaging.